The Heme Transport Capacity of LHR1 Determines the Extent of Virulence in Leishmania amazonensis

Abstract

Leishmania spp. are trypanosomatid parasites that replicate intracellularly in macrophages, causing serious human morbidity and mortality throughout the world. Trypanosomatid protozoa cannot synthesize heme, so must acquire this essential cofactor from their environment. Earlier studies identified LHR1 as a Leishmania amazonensis transmembrane protein that mediates heme uptake. Null mutants of LHR1 are not viable and single knockout strains have reduced virulence, but very little is known about the properties of LHR1 directly associated with heme transport. Here, we use functional assays in Saccharomyces cerevisiae to show that specific tyrosine residues within the first three predicted transmembrane domains of LHR1 are required for efficient heme uptake. These tyrosines are unique to LHR1, consistent with the low similarity between LHR1 and its corresponding homologs in C. elegans and human. Substitution of these tyrosines in LHR1 resulted in varying degrees of heme transport inhibition, phenotypes that closely mirrored the impaired ability of L. amazonensis to replicate as intracellular amastigotes in macrophages and generate cutaneous lesions in mice. Taken together, our results imply that the mechanism for heme transport by LHR1 is distinctive and may have adapted to secure heme, a limiting cofactor, inside the host. Since LHR1 is significantly divergent from the human heme transporter HRG1, our findings lay the groundwork for selective targeting of LHR1 by small molecule antagonists.

Trypanosomatid parasites rescue heme from endocytosed hemoglobin through lysosomal HRG transporters

“…This possibility remains to be proven, but the essential nature of TbHRG suggests that this putative alternative mechanism for heme scavenging also relies on the traffic of any form of heme to the parasite's endolysosome, requiring TbHRG for heme export to the cytoplasm. LHR1 is also essential for L. amazonensis (Huynh et al, 2012) (and probably for L. major) and required for the parasite's virulence in animal models (Miguel et al, 2013;Renberg et al, 2015). Although we have not analyzed the role of the syntenic HRG gene of T. cruzi, an essential role could also be expected for TcHRG, as T. cruzi, responsible for Chagas disease, is also auxotrophic for heme and performs Hb endocytosis (Lara et al, 2007).…”

“…Whether lysosomal LHR1 is physiologically relevant or is a product of its forced overexpression remains to be seen. Besides, LHR1 is essential for the parasite, and even the ablation of one allele strongly prevents the virulence of the parasite in animal models of cutaneous leishmaniasis, underlining its potential as a new drug target (Huynh et al, 2012;Miguel et al, 2013;Renberg et al, 2015).…”

The Trypanosoma brucei TbHrg protein is a heme transporter involved in the regulation of stage-specific morphological transitions

“…Heme deprivation in these protists increased the amount of Lhr1 mRNA 4-fold, which to a limited extent mimics the situation in C. elegans (22). Several unique amino acids were identified to be critical for the function of Lhr1, as their mutations affected transport of heme across cellular membranes and consequently the virulence of L. amazonensis (24). Recently, based on RNAi evidence, TbHrg was reported to be essential in BS of T. brucei and was characterized as a lysosomal transporter responsible for heme salvage from hemoglobin, downstream of HpHb receptor (HpHbR)-mediated import (25).…”

Trypanosoma cruzisenses intracellular heme levels and regulates heme homeostasis by modulatingTcHTE protein expression

“…During the last years, several proteins involved in heme transport have been described in trypanosomatids, such as LHR1 in Leishmania spp. (12)(13)(14), TbHRG in Trypanosoma brucei (16,17), and TcHTE in Trypanosoma cruzi (15). And most recently a protein identified in L. major, named LmFLVCRb and member of a Major Facilitator Superfamily, was described as a heme importer in this organism (23).…”

“…LHR1 mRNA responds to heme availability in the environment, confirming it as a member of the HRG family (12). Also, heme uptake mediated by LHR1 results relevant for the virulence of L. amazonensis (13,14). We described a homologous protein in T. cruzi, named TcHTE (T. cruzi Heme Transport Enhancer), that plays a critical role in heme transport and is found mainly in the flagellar pocket of T. cruzi (15).…”